The luminescent protein aequorin originates from the circumoral ring of the hydromedusa Aequorea victoria. When aequorin is bound to luciferin, it emits light on addition of Ca.sup.++. The binding of Ca.sup.++ to the aequorin-luciferin complex causes oxidation of the bound luciferin (an imidazopyrazine) yielding light (lambda max=470 nm) and carbon dioxide. This rapid oxidation allows for the detection of amols (10.sup.-18) of photoprotein (Campbell, A. K., et al., Methods of Biochemical Analysis 31:317-416, J. Wiley & Sons, Inc., London (1985)). The ability to detect this protein (and other proteins such as luciferase) at low concentrations, and its utility in a number of assays (Campbell, A. K., et al., Methods of Biochemical Analysis, 31:317-416, J. Wiley & Sons, Inc., London (1985)), suggests that a chimeric protein possessing specific affinity for analytes of interest as a chimeric protein possessing epitopes of an analyte (either immunoglobulins or antigens or portions thereof), and incorporating a photoprotein, would be of great value in immunoassay systems.
A chimeric gene is one comprising DNA or RNA genetic sequences from more than one source. The continuous polypeptide sequence or protein resulting from the expression of a chimeric genetic sequence is referred to here as a chimeric protein. A chimeric immunoglobulin is an immunoglobulin in which one or more of the subchains is a chimeric protein.
Chimeric immunoglobulins have been produced by the standard methods of genetic engineering. They may contain as an integral part of their structure enzymes and other biologically active peptides. Neuberger, M. S., et al., Nature 312:604-612 (1984); Neuberger, M. S., Trends in Biochemical Science, 347-349 (1985), Morrison, Science 229:1202 (1985). Chimeric antibodies have been produced which incorporate protein toxins, such as ricin, for selective destruction of specific target cells. PCT Patent Application PCT/GB85/00392, European Patent Application 120,694. Chimeric immunoglobulins have also been constructed which contain, as the incorporated genetic sequence, a section of another immunoglobulin gene from the same species. Sharon, J., et al., Nature 309:54 (1984). Immunoglobulin genetic sequences originating in different species have also been created and expressed as protein product. Oi and Morrison, Biotechniques 4:214 (1986). However, no immunoglobulin incorporating a photoprotein as an integral part of a chimeric protein structure is disclosed by the prior art.
Immunoglobulins tagged with photoproteins by conventional, i.e., nongenetic, biochemical linking methods have the utility of antibodies labeled with light emitting compounds and provide a sensitive and specific method of detecting an analyte of interest. U.K. Patent Application GB/2008247, European Patent Application 0137 515, U.S. Pat. No. 4,604,364. While immunoglobulins so tagged have proved useful, conventional methods of attaching photoproteins to immunoglobulins and antigens are difficult to perform and often lead to inactivation of the protein. Steric interference caused by the fused protein may also interfere with the proper binding of the immunoglobulin.
The genetic sequence of aequorin photoprotein is known (Inouye et al., Proc. Nat. Acad. Sci. 82:3154-3158 (1985)) and techniques have been disclosed to obtain the aequorin genetic sequence in a cloning vector (European Patent Application 0 187 519, Inouye et al., Biochem. 25:8425-8429 (1986)). It could not be predicted, however, whether a chimeric protein created by these techniques and containing aequorin would have the desirable luminescent properties of aequorin or any specific and desirable property of the immunoglobulin.
Both immunoglobulins and photoproteins depend for their proper functioning on their 3-dimensional structure. Such a 3-dimensional (tertiary) structure is frequently sensitive to and disrupted by changes in amino acid sequence (Tsuji, F. T., et al., Proc. Nat'l Acad. Sci. U.S.A. 83:8107-8111 (1986)). It could not be expected that the insertion of a genetic sequence for a photoprotein into the midst of the sequence of an immunoglobulin chain would produce a chimeric immunoglobulin which would properly function as desired. Similarly, in other chimeric photoproteins, such as chimeric photoprotein-antigens, changes in 3-dimensional folding in the chimeric protein could interfere or modify the properties of the chimeric protein in unexpected ways. Vora, S., Analytical Biochem. 144:307-318.